DNA microarrays in clinical cancer research

The recent sequencing of the human genome, coupled with advances in biotechnology, is enabling the comprehensive molecular "profiling" of human tissues. In particular, DNA microarrays are powerful tools for obtaining global views of human tumor gene expression. Complex information from tumor "expression profiling" studies can, in turn, be used to create novel molecular cancer diagnostics. We discuss the utility of DNA microarray-based tumor profiling in clinical cancer research, highlight some important recent studies, and identify future avenues of research in this evolving field.     

糖芯片技术在肿瘤研究中的应用

糖基化修饰是蛋白质常见的翻译后修饰之一,通过与糖结合蛋白如凝集素、抗体等相互作用调节肿瘤细胞侵袭、转移的能力及肿瘤异质性。通过化学合成法、化学-酶合成法或释放天然聚糖构建的糖芯片是分析聚糖与糖结合蛋白相互作用的重要工具。文中综述了常见的点制糖芯片的技术及糖芯片在癌症疫苗、单克隆抗体及诊断标志物中的广泛运用。由于肿瘤发生的各个环节都伴随着聚糖结构的改变,利用糖芯片探究肿瘤细胞特异表达的聚糖所参与的生理病理过程具有重大意义。     

The application of single nucleotide polymorphism microarrays in cancer research

The development of microarray technology has had a significant impact on the genetic analysis of human disease. The recently developed single nucleotide polymorphism (SNP) array can be used to measure both DNA polymorphism and dosage changes. Our laboratory has applied SNP microarray analysis to uncover frequent uniparental disomies and sub-microscopic genomic copy number gains and losses in different cancers. This review will focus on the wide range of applications of SNP microarray analysis to cancer research. SNP array genotyping can determine loss of heterozygosity, genomic copy number changes and DNA methylation alterations of cancer cells. The same technology can also be used to investigate allelic association in cancers. Therefore, it can be applied to the identification of cancer predisposition genes, oncogenes and tumor suppressor genes in specific types of tumors. As a consequence, they have potential in cancer risk assessment, diagnosis, prognosis and treatment selection.     

Protein microarrays and their applications

In recent years, the importance of proteomic works, such as protein expression, detection and identification, has grown in the fields of proteomic and diagnostic research. This is because complete genome sequences of humans, and other organisms, progress as cellular processing and controlling are performed by proteins as well as DNA or RNA. However, conventional protein analyses are time-consuming; therefore, high throughput protein analysis methods, which allow fast, direct and quantitative detection, are needed. These are so-called protein microarrays or protein chips, which have been developed to fulfill the need for high-throughput protein analyses. Although protein arrays are still in their infancy, technical development in immobilizing proteins in their native conformation on arrays, and the development of more sensitive detection methods, will facilitate the rapid deployment of protein arrays as high-throughput protein assay tools in proteomics and diagnostics. This review summarizes the basic technologies that are needed in the fabrication of protein arrays and their recent applications.     

Cell-biological applications of transfected-cell microarrays

Cell microarrays are a recent addition to the set of tools available for functional genomic studies. Each cell microarray is a slide with thousands of cell clusters that are each transfected with a defined DNA, which directs either the overproduction or the inhibition of a particular gene product. By using a range of detection assays, the phenotypic consequences of perturbing each gene in mammalian cells can be probed in a systematic, high-throughput fashion. Combining well-established methods for cellular investigation with the miniaturization and multiplexing capabilities of microarrays, cell arrays are a versatile tool that can be useful in many cell-biological applications.     

High-throughput proteomics using antibody microarrays

Antibody-based microarrays are a novel technology that hold great promise in proteomics. Microarrays can be printed with thousands of recombinant antibodies carrying the desired specificities, the biologic sample (e.g., an entire proteome) and any specifically bound analytes detected. The microarray patterns that are generated can then be converted into proteomic maps, or molecular fingerprints, revealing the composition of the proteome. Using this tool, global proteome analysis and protein expression profiling will thus provide new opportunities for biomarker discovery, drug target identification and disease diagnostics, as well as providing insights into disease biology. Intense work is currently underway to develop this novel technology platform into the high-throughput proteomic tool required by the research community.     

Secretome profiling with antibody microarrays

Following the advances in human genome sequencing, attention has shifted in part toward the elucidation of the encoded biological functions. Since proteins are the driving forces behind very many biological activities, large-scale examinations of their expression variations, their functional roles and regulation have moved to the central stage. A significant fraction of the human proteome consists of secreted proteins. Exploring this set of molecules offers unique opportunities for understanding molecular interactions between cells and fosters biomarker discovery that could advance the detection and monitoring of diseases. Antibody microarrays are among the relatively new proteomic methodologies that may advance the field significantly because of their relative simplicity, robust performance and high sensitivity down to single-molecule detection. In addition, several aspects such as variations in amount, structure and activity can be assayed at a time. Antibody microarrays are therefore likely to improve the analytical capabilities in proteomics and consequently permit the production of even more informative and reliable data. This review looks at recent applications of this novel platform technology in secretome analysis and reflects on the future.     

Immunostaining with dissociable antibody microarrays

The availability of a large number of biological materials such as cDNA, antibodies, recombinant proteins, and tissues has promoted the development of microarray technologies that make use of these materials in high-throughput screening assays. However, because microarray technologies have been less successful in examining proteins than DNA and mRNA, there is a need for improved protein microarray systems. To address this need, we developed an antibody microarray-based immunostaining method that can analyze the properties of a large number of proteins simultaneously. In this method, antibodies are arrayed and immobilized on a solid support and cells bearing antigens of interest are attached to a second support. Apposition of the two supports allows the antibodies to dissociate from the array support and bind to the cellular antigens. After separation of the supports, antigen-bound antibodies can be detected by standard secondary antibody techniques. These "dissociable" antibody arrays were used to detect both the expression and subcellular localization of a large number of specific proteins in various cultured cell types.     

High-throughput proteomics using antibody microarrays

Antibody-based microarrays are a novel technology that hold great promise in proteomics. Microarrays can be printed with thousands of recombinant antibodies carrying the desired specificities, the biologic sample (e.g., an entire proteome) and any specifically bound analytes detected. The microarray patterns that are generated can then be converted into proteomic maps, or molecular fingerprints, revealing the composition of the proteome. Using this tool, global proteome analysis and protein expression profiling will thus provide new opportunities for biomarker discovery, drug target identification and disease diagnostics, as well as providing insights into disease biology. Intense work is currently underway to develop this novel technology platform into the high-throughput proteomic tool required by the research community.     

Diagnostic and analytical applications of protein microarrays

DNA microarrays have changed the field of biomedical sciences over the past 10 years. For several reasons, antibody and other protein microarrays have not developed at the same rate. However, protein and antibody arrays have emerged as a powerful tool to complement DNA microarrays during the past 5 years. A genome-scale protein microarray has been demonstrated for identifying protein–protein interactions as well as for rapid identification of protein binding to a particular drug. Furthermore, protein microarrays have been shown as an efficient tool in cancer profiling, detection of bacteria and toxins, identification of allergen reactivity and autoantibodies. They have also demonstrated the ability to measure the absolute concentration of small molecules. Besides their capacity for parallel diagnostics, microarrays can be more sensitive than traditional methods such as enzyme-linked immunosorbent assay, mass spectrometry or high-performance liquid chromatography-based assays. However, for protein and antibody arrays to be successfully introduced into diagnostics, the biochemistry of immunomicroarrays must be better characterized and simplified, they must be validated in a clinical setting and be amenable to automation or integrated into easy-to-use systems, such as micrototal analysis systems or point-of-care devices.     

Diagnostic and analytical applications of protein microarrays

DNA microarrays have changed the field of biomedical sciences over the past 10 years. For several reasons, antibody and other protein microarrays have not developed at the same rate. However, protein and antibody arrays have emerged as a powerful tool to complement DNA microarrays during the past 5 years. A genome-scale protein microarray has been demonstrated for identifying protein-protein interactions as well as for rapid identification of protein binding to a particular drug. Furthermore, protein microarrays have been shown as an efficient tool in cancer profiling, detection of bacteria and toxins, identification of allergen reactivity and autoantibodies. They have also demonstrated the ability to measure the absolute concentration of small molecules. Besides their capacity for parallel diagnostics, microarrays can be more sensitive than traditional methods such as enzyme-linked immunosorbent assay, mass spectrometry or high-performance liquid chromatography-based assays. However, for protein and antibody arrays to be successfully introduced into diagnostics, the biochemistry of immunomicroarrays must be better characterized and simplified, they must be validated in a clinical setting and be amenable to automation or integrated into easy-to-use systems, such as micrototal analysis systems or point-of-care devices.     

Fabrication and characterization of 3D hydrogel microarrays to measure antigenicity and antibody functionality for biosensor applications

We report the fabrication, characterization and evaluation of three-dimensional (3D) hydrogel thin films used to measure protein binding (antigenicity) and antibody functionality in a microarray format. Protein antigenicity was evaluated using the protein toxin, staphylococcal enterotoxin B (SEB), as a model on highly crosslinked hydrogel thin films of polyacrylamide and on two-dimensional (2D) glass surfaces. Covalent crosslinking conditions were optimized and quantified. Interrogation of the modified 3D hydrogel was measured both by direct coupling of a Cy5-labeled SEB molecule and Cy5-anti-SEB antibody binding to immobilized unlabeled SEB. Antibody functionality experiments were conducted using three chemically modified surfaces (highly crosslinked polyacrylamide hydrogels, commercially available hydrogels and 2D glass surfaces). Cy3-labeled anti-mouse IgG (capture antibody) was microarrayed onto the hydrogel surfaces and interrogated with the corresponding Cy5-labeled mouse IgG (antigen). Five different concentrations of Cy5-labeled mouse IgG were applied to each microarrayed surface and the fluorescence quantified by scanning laser confocal microscopy. Experimental results showed fluorescence intensities 3-10-fold higher for the 3D films compared to analogous 2D surfaces with attomole level sensitivity measured in direct capture immunoassays. However, 2D surfaces reported equal or greater sensitivity on a per-molecule basis. Reported also are the immobilization efficiencies, inter-and intra-slide variability and detection limits.     

CGH microarrays and cancer

Genetic alterations are a key feature of cancer cells and typically target biological processes and pathways that contribute to cancer pathogenesis. Array-based comparative genomic hybridization (aCGH) has provided a wealth of new information on copy number changes in cancer on a genome-wide level and aCGH data have also been utilized in cancer classification. More importantly, aCGH analyses have allowed highly accurate localization of specific genetic alterations that, for example, are associated with tumor progression, therapy response, or patient outcome. The genes involved in these aberrations are likely to contribute to cancer pathogenesis, and the high-resolution mapping by aCGH greatly facilitates the subsequent identification of these cancer-associated genes.